Gram-negative pathogens are becoming increasingly resistant to currently used antimicrobials. Furthermore, the pipeline for new antibiotics is slim and new therapies are urgently needed. This can be especially problematic in patients who suffer from chronic infections. Examples of inherently drug resistant Gram-negative pathogens are Pseudomonas aeruginosa and the Burkholderia cepacia complex (Bcc). These pathogens cause disease in a variety of settings, but are particularly devastating in patients with cystic fibrosis (CF). Chronic P. aeruginosa or Bcc infection can lead to both progressive pulmonary decline, as well as in Bcc infection, rapid deterioration in lung function referred to as cepacia syndrome. We have been interested in using antisense molecules called PPMOs as potential therapeutics in these infections. These molecules block messenger RNA and block the formation of protein. We have demonstrated that PPMOs can be used to target genes that are essential for these pathogens to grow, specifically the gene acpP (that encodes the protein acyl carrier protein). We showed that blocking this protein is essential for Burkholderia and Pseudomonas to grow in vitro. We also showed that this PPMO improves survival in mice that were infected with Burkholderia. For this project, we propose to find other essential gene targets in these pathogens that can be blocked with PPMOs, and perform studies to evaluate the most promising compounds ability to improve survival in mouse models of infection. In the first phase of the project, we will target pathways that have been shown in other Gram-negative pathogens to be essential for growth. These will include genes important in the development of the cell wall and associated structures (lipopolysaccharide and peptidoglycan biosynthesis) as well as fatty acid biosynthesis (i.e. acyl carrier protein). In addition, we will try to block the genes that pla a role in antibiotic resistance to see if we can restore activity of currently existing antibiotics. he second phase of the project will be to evaluate lead PPMO candidates in mouse models of infection. We will use the PPMOs as treatments in pulmonary models of infection, assessing both systemic and pulmonary delivery of the compound. By the conclusion of this study, we hope to have promising novel treatments for two of the most devastating pathogens in CF that could advance to further clinical development. This work has implications not only for these infections in CF, but for other medically important Gram-negative pathogens.